Establishment of CRFK cells for vaccine production by inactivating endogenous retrovirus with TALEN technology

Endogenous retroviruses (ERVs) are retroviral sequences present in the host genomes. Although most ERVs are inactivated, some are produced as replication-competent viruses from host cells. We previously reported that several live-attenuated vaccines for companion animals prepared using the Crandell-Rees feline kidney (CRFK) cell line were contaminated with a replication-competent feline ERV termed RD-114 virus. We also found that the infectious RD-114 virus can be generated by recombination between multiple RD-114 virus-related proviruses (RDRSs) in CRFK cells. In this study, we knocked out RDRS env genes using the genome-editing tool TAL Effector Nuclease (TALEN) to reduce the risk of contamination by infectious ERVs in vaccine products. As a result, we succeeded in establishing RDRS knockout CRFK cells (RDKO_CRFK cells) that do not produce infectious RD-114 virus. The growth kinetics of feline herpesvirus type 1, calicivirus, and panleukopenia virus in RDKO_CRFK cells differed from those in parental cells, but all of them showed high titers exceeding 107 TCID50/mL. Infectious RD-114 virus was undetectable in the viral stocks propagated in RDKO_CRFK cells. This study suggested that RDRS env gene-knockout CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with no risk of contamination with infectious ERV.

www.nature.com/scientificreports/ ERV is activated and induces lymphoma 17,18 . Additionally, two independent research groups reported that infectious murine ERVs could exhibit oncogenicity in mice 19,20 . In their studies, replication-competent ERVs were regenerated in immunodeficient mice by recombination between replication-defective ERVs, and the regenerated ERVs induced lymphoma in the hosts. Similar to cases in mice, once the infectious RD-114 virus is regenerated in cats, it may re-infect a wide variety of feline tissues in vivo. The pathogenic potential of the RD-114 virus is unknown at present; however, RD-114 viral expression in domestic cats' lymphoma and malignant tissues is significantly higher than in normal tissues 21,22 , and a large granular lymphoma cell line, MCC cells, produces infectious RD-114 virus 14 . RD-114 virus also productively infects primary canine epithelial cells as well as canine cell lines 23 . RD-114 viral receptor ASCT2 is also expressed ubiquitously in canine tissues 24 . Therefore, RD-114 virus potentially infects both cats and dogs in vivo.
In the domestic cat genome, there are several copies of RD-114 virus-related sequences (RDRSs) which correspond to a group of ERVs 9,25 . Previously, we identified loci and sequences of six RDRSs 26 . All domestic cats have only one RDRS, located on chromosome C2 and referred to as RDRS C2a, but populations of the other five RDRS s are different depending on the regions where the cats live or breed. Although RDRS C2a has been present in the domestic cat genome for millions of years, its function remains unknown. We demonstrated that infectious RD-114 virus might have been generated by recombination between two RDRSs (RDRS A2 [RDRS on chromosome A2] and RDRS C1 [RDRS on chromosome C1]) in CRFK cells 26 . CRFK cells also have RDRS C2a, but it has stop codons within all viral genes and cannot be the source of infectious RD-114 virus. In this study, to eliminate infectious RD-114 viral particles, we targeted RDRS env genes and used the genome-editing tool TAL Effector (TALE) Nuclease (TALEN), which can destroy multiple targets. TALEN consists of DNA-binding domain, TALE, and FokI nuclease domain. The DNA-binding repeat of TALE is composed of tandem 34-amino acid modules, each of which recognizes a single base of DNA 27 . The most widely used genome editing tool, CRISPR-Cas9, is simple to design and use, requiring only 20 bases of target sites to be programmed 28 . However, TALEN remains valuable due to its relatively unconstrained target site and high specificity. The target sequence of CRISPR-Cas9 must be immediately followed by a proto-spacer adjacent motif (PAM) sequence. This target constraint can make it challenging to design and edit targets. Regarding target specificity, CRISPR-Cas9 has been reported to have off-targets that cleave sequences different from the target sequence 29 . TALEN has fewer off-targets because it is a cleavage scheme that induces double-strand breaks only when a pair of TALENs binds correctly to the target sequence, and the target sequence is longer than CRISPR-Cas9. In this study, we chose to use Platinum TALEN, a highly active type of TALEN 30 . Platinum TALEN has been shown to have high cleavage activity in a variety of cells and organisms, including nematodes, sea urchins, newts, frogs, mice, and rats 31 .

Results and discussion
CRFK cells produce infectious RD-114 virus 10 . The infectious RD-114 virus can be regenerated by recombination between two RDRSs (RDRS A2 and C1) in CRFK cells 26 . To prevent the generation of infectious RD-114 virus by recombination, we aimed to knock out env genes of RDRS A2 and C1 using TALEN technology, which can destroy multiple targets. Firstly, we designed the TALEN-targeting site on the surface unit (SU) domain of env genes (Fig. 1). SU is responsible for binding to a specific receptor on target cells, which is necessary for retroviral infection. We transfected CRFK cells with TALEN plasmids. pcDNA3.1 was used as a negative control. Two days after transfection, genomic DNA was extracted and screened for TALEN-induced mutations using the Surveyor (Cel-I) nuclease assay. Cel-I nuclease recognizes and cuts mismatched heteroduplex DNA formed between wildtype and mutant DNA 32 . TALEN pair-B but not pair-A showed high mutation efficiency (Fig. 2a). Sequencing analysis of RDRS env genes revealed that two out of 10 clones contained deletions at the targeted site (Fig. 2b). To generate CRFK cell clones in which env genes of both RDRS A2 and C1 were disrupted, we co-transfected CRFK cells with TALEN pair-B plasmid with an expression plasmid of the puromycin-resistant gene. To select cells expressing transfected DNA, a selection marker needs to be co-expressed on either the same construct or on a separate vector that is co-transfected into the cell. In this study, we chose a method to co-transfect with a puromycin-resistant gene plasmid for selection. The TALEN plasmid contains a hygromycin-resistant gene, however, CRFK cells are relatively resistant to hygromycin, making it difficult to select TALEN-transduced cells with hygromycin. Therefore, we cotransfected the puromycin-resistant gene plasmid with TALEN plasmid. CRFK cells are highly sensitive to puromycin, making it easy to select TALEN-transduced cells. Most of the puromycin-resistant clones were also transduced with the TALEN plasmid. One month after puromycin selection, 31 clones were picked up and screened by PCR of genomic DNA and sequencing. In these clones, only one clone was identified as homozygous env gene knockout cells on both RDRSs. This clone was re-cloned to ensure clonality, and we named the clone RDKO_CRFK cells. This clone carries different mutations on each allele of the env gene of RDRS A2 and C1 (Fig. 3). To confirm whether RDRS env disruption results in the inhibition of RD-114 viral production, we cultured parental CRFK and RDKO_CRFK cells for three days and measured the copy numbers of RDRS env RNA in the culture supernatants by quantitative real-time RT-PCR ( Fig. 4a,b). As a result, we could not detect RDRS env RNA in the culture supernatant of RDKO_CRFK cells (Fig. 4b). The S+L− focus assay indicated that the infectious titer of RD-114 virus produced from RDKO_CRFK cells was below the detection limit (Fig. 4c). We have passaged RDKO_CRFK cells four times to investigate if they produced RD-114 virus. When a long-term culture is performed, the defective RDRSs may cause recombination again if it transcribed in RDKO_CRFK cells. However, we have confirmed that there are only three copies of env genes in the CRFK cell genome in previous report 26 and that all of them have been disrupted; env gene of RDRS C2a also disrupted in RDKO_CRFK cells ( Supplementary Fig. S2). Therefore, there is no possibility of producing RD-114 virus with a full-length env gene. The cell viability of RDKO_CRFK cells was similar to that of parental CRFK cells, indicating that the env knockout does not affect the proliferation of CRFK cells (Fig. 4d). www.nature.com/scientificreports/ CRFK cells have been used to propagate several viruses for vaccines for animals, including core feline trivalent inactivated vaccines called "FVRCP vaccines" against feline herpesvirus type 1 (FHV-1), calicivirus (FCV), and panleukopenia virus (FPLV). We examined the effects of env knockout on the growth of FHV-1, FCV, and FPLV. Parental CRFK and RDKO_CRFK cells were inoculated with FHV-1, FCV, and FPLV and cultured for 4, 2, and 6 days, respectively, and the titer (expressed as TCID 50 ) of each virus was measured. The titer of FHV-1 in RDKO_CRFK cells was similar in the parental cells, but the titer of FCV was significantly lower and that of FPLV was significantly higher (Fig. 5a). These results indicate that the expression of RDRS had some effect on FHV-1, FCV and FPLV growth. Further analyses, for this reason, would be required. We also measured the copy number of RDRS env RNA in the viruses produced in both parental CRFK and RDKO_CRFK cells by real-time RT-PCR. The copy numbers of RDRS env RNA in the preparation of FHV-1, FCV, and FPLV from RDKO_CRFK cells were below detection limits (Fig. 5b).
Finally, to investigate the potential off-target effects of TALEN, we first searched the reference sequence Felis_catus_8.0 (GenBank accession number: GCA_0001811335.3) for sites that shared similar sequences with TALEN pair-B target. The top ten potential off-target sites were sequenced. None of the sequences of off-target candidates were mutated (Table 1).
There are several cases of ERV-contaminated vaccines for not only companion animals but also humans. MMR vaccines and yellow fever vaccines grown in chicken embryo fibroblasts were contaminated with endogenous ALVs and endogenous EAVs, originating from chicken embryonic fibroblast substrates 2,3 . Herpes virus 3 vaccine grown in human embryonic lung cells was contaminated with human ERV K 33 . Our results may be useful for the manufacture of live-attenuated vaccines with no risk of contamination with infectious ERVs. Recently, it was reported that all 62 copies of the porcine endogenous retroviruses (PERVs) were disrupted in a pig kidney epithelial cell line by CRISPR-Cas9 mediated genome editing 34 and generated PERV-inactivated pigs via somatic cell nuclear transfer 35 . Our strategy to control ERV production as well as the method of PERV knockout, can be used to treat other cell lines for vaccine production, but also for studies of the function of ERVs.     Fig. 1. Cel-I assay and sequencing. Two days after transfection, genomic DNA was extracted from CRFK cells using QIAamp DNA Blood Mini kit (QIAGEN, CA, USA). PCR was performed using PrimeSTAR GXL polymerase (TaKaRa, Shiga, Japan) according to the manufacturer's instructions. Primers used for this PCR were listed in Table 2. The amplicons were heat-denatured, digested by Surveyor nuclease (Transgenomic, NE, USA), and subjected to agarose gel electrophoresis to detect TALEN-induced mutations.

Knockout of RDRS env in CRFK cells. CRFK cells (5 × 10
The PCR products were also cloned into pCR BluntII TOPO plasmid vector (Life Technologies, CA, USA) and sequenced. Sequencing was performed by a commercial DNA sequencing service (FASMAC, Kanagawa, Japan). Alignment of nucleotide sequences and estimation of homology were performed using GENETYX win Ver.11 (GENETYX, Tokyo, Japan).

Selection of TALEN-transfected CRFK cells. TALEN-transfected CRFK cells were seeded in 10-cm
dishes and selected with 4 μg/mL of puromycin (InvivoGen, CA, USA). Then, AH927 cells, which do not contain RDRS A2 or C1 in their genomes (thus, AH927 cells are RD-114 non-producer cells), were co-cultured as www.nature.com/scientificreports/ feeder cells to supply the nutrients for the small colonies. Medium with puromycin was replaced every 3 days. One month after starting selection with puromycin, individual puromycin-resistant cell colonies were picked up and cultured in 96-multiwell plates. Two days after transferring the cells into 96-multiwell plates, the cells were further subcultured in 6-multiwell plates. The clones were then subjected to PCR and sequencing analyses using genomic DNA.  Table 2.

Titration of infectious RD-114 virus.
Replication-competent gammaretrovirus in the culture supernatants of parental CRFK and RDKO_CRFK cells was evaluated by the S+L− assay using QN10S cells 39 . QN10S cells were seeded in 24-well plates at 1 × 10 4 cells/well one day before infection and diluted supernatants of RDKO_CRFK cells were used to inoculate QN10S cells in the presence of polybrene (Sigma-Aldrich) (8 µg/mL). Seven days after inoculation, numbers of foci were counted.
Cell proliferation assay. Cell proliferations of parental CRFK and RDKO_CRFK cells were measured using Cell Proliferation Kit I (MTT) (Roche Diagnostics). The cells were seeded at 1 × 10 5 cells/well in 96-multiwell plates and then cultured for 2 days. The assay was performed according to the manufacturer's instructions. Infection and titration of feline herpesvirus type 1, calicivirus, and panleukopenia virus. FHV-1 strain 00-015 40 , FCV strain 01-106, and FPLV strain V142 41 were used. To evaluate the growth kinetics of FHV-1 strain 00-015, 1000-fold dilutions of a virus stock are prepared, and 60 µL aliquots are used to inoculate parental CRFK and RDKO_CRFK cell monolayers in 6-multiwell plates. The cells were inoculated with FCV strain 01-106 at a multiplicity of infection (MOI) of 0.01 and FPLV strain V142 at MOI of 0.1. After incubation at 37 °C for 1 h, the cells were washed three times with PBS and then cultured with 2 mL of DMEM supplemented with 10% FCS for 4, 2, and 6 days, respectively. The titers of FHV-1 and FCV produced in parental CRFK or RDKO_ CRFK cells were measured in TCID 50 using CRFK cells, as described previously 40,42 . The titers of FPLV produced in parental CRFK or RDKO_CRFK cells were measured in TCID 50 using FL74 cells, as described previously 43,44 .